High-fidelity DNA replication depends on a proofreading 3′–5′ exonuclease that is associated with the replicative DNA polymerase. The replicative DNA polymerase DnaE1 from the major pathogen Mycobacterium tuberculosis (Mtb) uses its intrinsic PHP-exonuclease that is distinct from the canonical DEDD exonucleases found in the Escherichia coli and eukaryotic replisomes. The mechanism of the PHP-exonuclease is not known. Here, we present the crystal structure of the Mtb DnaE1 polymerase. The PHP-exonuclease has a trinuclear zinc center, coordinated by nine conserved residues. Cryo-EM analysis reveals the entry path of the primer strand in the PHP-exonuclease active site. Furthermore, the PHP-exonuclease shows a striking similarity to E. coli endonuclease IV, which provides clues regarding the mechanism of action. Altogether, this work provides important insights into the PHP-exonuclease and reveals unique properties that make it an attractive target for novel anti-mycobacterial drugs.

The conserved Ca2+-binding protein Frequenin/Neuronal Calcium Sensor 1 (Frq/NCS-1) is involved in pathologies that result from abnormal synapse number and probability of neurotransmitter release per synapse. Both synaptic features are likely co-regulated but the intervening mechanisms remain poorly understood. We show here that Drosophila Ric8a/Synembryn, a receptor-independent activator of G protein complexes, binds Frq2 but not the virtually identical duplicate Frq1. Based on crystallographic data on Frq2 and site directed mutagenesis on Frq1, the differential amino acids R94 and T138 account for this specificity. Human NCS-1 and Ric8a reproduce the binding and maintain the structural requirements at these key positions. Ric8a and Gαs regulate synapse number and neurotransmitter release and both are functionally linked to Frq2. Frq2 negatively regulates Ric8a to control synapse number. However, the regulation of neurotransmitter release by Ric8a is independent from the Frq2 binding. Thus, the antagonistic regulation of these two synaptic properties shares a common pathway, Frq2,Ric8a,Gαs which diverges downstream. These mechanisms expose the Frq2-Ric8a interacting surface as a potential pharmacological target for NCS-1 related diseases and provide key data towards the corresponding drug design.

The endolysosomal system is a highly dynamic network of membranes for degradation and recycling. During endosomal maturation, cargo molecules destined for lysosomal degradation are progressively concentrated through continuous rounds of fusion and fission reactions concomitant with inbound and outbound membrane fluxes. Of the cargo molecules delivered to endosomes, about two-thirds are rescued from degradation and recycled for reuse. This balance between degradation and recycling is essential to preserve the proteostatic plasticity of the cell under variable physiological demands. Cargo retrieval from endosomes involves several sorting complexes with stable core compositions that associate with multidomain regulatory proteins, consequently displaying complex interaction networks. The vacuolar protein sorting 29 (VPS29) has emerged as a central scaffold that coordinates the physical assembly of retrieval complexes with regulatory components in what appears to be an elegant solution for regulating distinct retrieval stations. This review summarizes the VPS29-binding partners and its integration into retrieval complexes for endosomal sorting and trafficking.

Transduction of producer cells during lentiviral vector (LVV) production causes the loss of 70–90% of viable particles. This process is called retro-transduction and it is a consequence of the interaction between the LVV envelope protein, VSV-G, and the LDL receptor located on the producer cell membrane, allowing lentiviral vector transduction. Avoiding retro-transduction in LVV manufacturing is crucial to improve net production and, therefore, the efficiency of the production process. Here, we describe a method for quantifying the transduction of producer cells and three different strategies that, focused on the interaction between VSV-G and the LDLR, aim to reduce retro-transduction.

Background/Objectives: To date, nearly 300 lentiviral-based gene therapy clinical trials have been conducted, with eight therapies receiving regulatory approval for commercialization. These advances, along with the increased number of advanced-phase clinical trials, have prompted contract development and manufacturing organizations (CDMOs) to develop innovative strategies to address the growing demand for large-scale batches of lentiviral vectors (LVVs). Consequently, manufacturers have focused on optimizing processes under good manufacturing practices (GMPs) to improve cost-efficiency, increase process robustness, and ensure regulatory compliance. Nowadays, the LVV production process mainly relies on the transient transfection of four plasmids encoding for the lentiviral helper genes and the transgene. While this method is efficient at small scales and has also proven to be scalable, the industry is exploring alternative processes due to the high cost of GMP reagents, and the batch-to-batch variability predominantly attributed to the transfection step. Methods: Here, we report the development and implementation of a reliable and clinical-grade envisioned platform based on the generation of stable producer cell lines (SCLs) from an initial well-characterized lentiviral packaging cell line (PCL). Results: This platform enables the production of VSV-G-pseudotyped LVVs through a fully transfection-free manufacturing process. Our data demonstrate that the developed platform will facilitate successful technological transfer to large-scale LVV production for clinical application. Conclusions: With this simple and robust stable cell line generation strategy, we address key concerns associated with the costs and reproducibility of current manufacturing processes.

Extensive studies about the human immunodeficiency virus type 1 (HIV-1) have allowed the generation of lentiviral vectors as gene delivery vehicles with enhanced safety and efficacy features. In this review, several strategies for controlling the molecular mechanisms occurring during the lentiviral vector manufacturing process are presented. Specifically, modifications focused on LVV manufacturing components, such as plasmids or the producer cell line, that enable increased safety, integrity, and potency of the produced LVV, as well as manufacturing efficiency. Considering the stochasticity of the LVV manufacturing process from plasmid transfection until the budding of the virus from the target cell, minimal modifications might have a huge impact on the final LVV yield. Indeed, the extent of a potential impact may vary depending on the specificities of each LVV regarding the particular genetic payload or the envelope protein. Thus, the feasibility of each of the optimizations described herein requires thorough evaluation. The second part of the review examines the potential multi-purpose nature of the LVV. Growing research in the field has enabled the development of new engineered modalities of LVV, expanding their application scope beyond the traditional ex vivo DNA delivery approach. LVVs are becoming a versatile tool for the packaging or delivery of cargo in the form of DNA, RNA, or protein, allowing their use for in vivo approaches, vaccinology, or gene editing, among others.